Method for Measuring Dissociation Constant by Surface Plasmon Resonance Analysis

ABSTRACT

An object to be solved by the present invention is to determine the dissociation constant of an analyte molecule immobilized on a metal surface and a molecule that interacts therewith in surface plasmon resonance (SPR) analysis via an analytical method that produces a low noise level (i.e., a noise width of a reference chip), small baseline fluctuations (i.e., signal changes of a reference chip), and highly reliable results of measurement. The present invention provides a method for determining a dissociation constant of an analyte molecule immobilized on a metal surface and a molecule that interacts therewith, wherein changes in surface plasmon resonance signals are measured by using a surface plasmon resonance measurement device comprising a flow channel system having a cell formed on a metal film and a light-detecting means for detecting the state of surface plasmon resonance by measuring the intensity of a light beam totally reflected on the metal film; wherein a change in surface plasmon resonance signals is measured in a state where the flow of the liquid has been stopped, after the liquid contained in the above flow channel system has been exchanged; and wherein the dissociation constant is determined based on the results of measurement of signal changes.

TECHNICAL FIELD

The present invention relates to a method for measuring the dissociationconstant of an analyte molecule immobilized on a metal surface and amolecule that interacts therewith by surface plasmon resonance (SPR)analysis.

BACKGROUND ART

Recently, a large number of measurements using intermolecularinteractions such as immune responses are being carried out in clinicaltests, etc. However, since conventional methods require complicatedoperations or labeling substances, several techniques are used that arecapable of detecting the change in the binding amount of a testsubstance with high sensitivity without using such labeling substances.Examples of such a technique may include a surface plasmon resonance(SPR) measurement technique, a quartz crystal microbalance (QCM)measurement technique, and a measurement technique of using functionalsurfaces ranging from gold colloid particles to ultra-fine particles.The SPR measurement technique is a method of measuring changes in therefractive index near an organic functional film attached to the metalfilm of a chip by measuring a peak shift in the wavelength of reflectedlight, or changes in amounts of reflected light in a certain wavelength,so as to detect adsorption and desorption occurring near the surface.The QCM measurement technique is a technique of detecting adsorbed ordesorbed mass at the ng level, using a change in frequency of a crystaldue to adsorption or desorption of a substance on gold electrodes of aquartz crystal (device). In addition, the ultra-fine particle surface(nm level) of gold is functionalized, and physiologically activesubstances are immobilized thereon. Thus, a reaction to recognizespecificity among physiologically active substances is carried out,thereby detecting a substance associated with a living organism fromsedimentation of gold fine particles or sequences. Surface plasmonresonance (SPR), which is most commonly used in this technical field,will be described below as an example.

A commonly used measurement chip comprises a transparent substrate(e.g., glass), an evaporated metal film, and a thin film having thereona functional group capable of immobilizing a physiologically activesubstance. The measurement chip immobilizes the physiologically activesubstance on the metal surface via the functional group. A specificbinding reaction between the physiological active substance and a testsubstance is measured, so as to analyze an interaction betweenbiomolecules. An example of a surface plasmon resonance measurementdevice is the device described in Japanese Patent Laid-Open (Kokai) No.2001-330560.

When a specific binding reaction between a physiologically activesubstance and a test substance is measured, the binding reaction isgenerally measured by: connecting in series a reference cell, to which aphysiologically active substance interacting with a test substance doesnot bind, with a detection cell, to which a physiologically activesubstance interacting with a test substance binds; placing the connectedcells in a flow channel system; and feeding a liquid through thereference cell and the detection cell, so as to carry out themeasurement of the binding reaction. During the measurement, the liquidcontained in the above flow channel system is exchanged from a referenceliquid containing no test substance to be measured to a sample liquidcontaining a test substance to be measured, so as to cause the bindingreaction between the physiologically active substance and the testsubstance to be initiated, and to measure a change in signals due to alapse of time.

As mentioned above, a biosensor that uses SPR detects the binding of ananalyte to a sensor (a metal film and a ligand) as a change in therefractive index (and an angular change of a dark line causedthereupon). If the time is plotted on the horizontal axis and thebinding signal is plotted on the vertical axis, a signal (the amount ofbinding or the like) that is referred to as a so-called “sensorgram” canbe observed with the elapse of time. It is important to carry outfitting of the following rate equation (i) to the sensorgram followed bydetermination of the rate coefficients such as the adsorption ratecoefficient (ka) and the dissociation rate coefficient (kd).dR/dt=ka×C×{R max−R(t)}−kd×R(t)   (i)R(t)=(ka×C×R max)/(ka×C+kd)×(1−exp(−kax×C+kd)×t)) (ii) (the result ofsolving equation (i))wherein ka represents an adsorption rate coefficient; kd represents adissociation rate coefficient; C represents an analyte concentration(known); Rmax represents the theoretical maximum amount of binding; andt represents a time.

DISCLOSURE OF THE INVENTION

Object to be Solved by the Invention

According to the aforementioned method for measuring signal changes withthe elapse of time, wherein the liquid contained in the above flowchannel system is exchanged from a reference liquid containing no testsubstance to be measured to a sample liquid containing a test substanceto be measured, so as to cause the binding reaction between thephysiologically active substance and the test substance to be initiated,and to measure a change in signals due to a lapse of time, a noise widthand baseline fluctuations of signal changes of a reference cell within ameasurement time were problematic, and it was difficult to attain highlyreliable data for the binding detection. As a means for dissolving suchdrawback, JP Patent Application No. 2003-404040 discloses a method formeasuring a change in surface plasmon resonance, which comprises: usinga surface plasmon resonance measurement device comprising a flow channelsystem having a cell formed on a metal film and a light-detecting meansfor detecting the state of surface plasmon resonance by measuring theintensity of a light beam totally reflected on the metal film; andexchanging the liquid contained in the above flow channel system,wherein the above method is characterized in that a change in surfaceplasmon resonance is measured in a state where the flow of the liquidhas been stopped, after the liquid contained in the above flow channelsystem has been exchanged. By measuring changes in surface plasmonresonance in a state where the flow of the liquid has been stopped,after the liquid contained in the above flow channel system has beenexchanged, highly reliable results of measurement with a low noise level(i.e., a noise width of a reference chip) and small baselinefluctuations (i.e., signal changes of a reference chip) can be attained.However, there has been no report concerning the measurement of ratecoefficients such as ka (an adsorption rate coefficient) and kd (adissociation rate coefficient) based on the results of measurement ofchanges in SPR signals obtained by the method of measuring signalchanges in a state where the flow of the liquid has been stopped, afterthe liquid contained in the flow channel system of the surface plasmonresonance measurement device has been exchanged. Accordingly, an objectto be solved by the present invention is to determine the dissociationconstant of an analyte molecule immobilized on a metal surface and amolecule that interacts therewith in surface plasmon resonance (SPR)analysis via an analytical method that produces a low noise level (i.e.,a noise width of a reference chip), small baseline fluctuations (i.e.,signal changes of a reference chip), and highly reliable results ofmeasurement.

Means for Solving the Object

The present inventors have demonstrated for the first time that ratecoefficients such as ka (an adsorption rate coefficient) and kd (adissociation rate coefficient) could be accurately determined based onthe results of measurement of signal changes obtained by the method formeasuring SPR signal changes in a state where the flow of the liquid hasbeen stopped, after the liquid contained in the flow channel system ofthe surface plasmon resonance measurement device has been exchanged.This has led to the completion of the present invention.

Thus, the present invention provides a method for determining adissociation constant of an analyte molecule immobilized on a metalsurface and a molecule that interacts therewith, wherein changes insurface plasmon resonance signals are measured by using a surfaceplasmon resonance measurement device comprising a flow channel systemhaving a cell formed on a metal film and a light-detecting means fordetecting the state of surface plasmon resonance by measuring theintensity of a light beam totally reflected on the metal film; wherein achange in surface plasmon resonance signals is measured in a state wherethe flow of the liquid has been stopped, after the liquid contained inthe above flow channel system has been exchanged; and wherein thedissociation constant is determined based on the results of measurementof signal changes.

Preferably, an adsorption rate coefficient (ka) and a dissociation ratecoefficient (kd) are determined based on the results of measurement ofchanges in surface plasmon resonance signals, and a dissociationconstant (KD) is determined using an equation represented by KD=kd/kabased on the determined adsorption rate coefficient (ka) and thedissociation rate coefficient (kd).

Preferably, an adsorption rate coefficient (ka) and a dissociation ratecoefficient (kd) are determined based on the results of measurement ofchanges in surface plasmon resonance signals by using the followingequations (1), (2), and (3):dθ/dt=ka×c _(s)×(1−θ)−kd×0   (1)wherein θ represents an adsorption ratio (=amount of adsorption/amountof saturated adsorption); ka represents an adsorption rate coefficient;kd represents a dissociation rate coefficient; and c_(s) represents theconcentration of the analyte molecule that exists near the metalsurface;∂c/∂t=D×∂ ² c/∂x ²   (2)wherein x represents a distance from a metal surface; D represents adiffusion coefficient of the analyte molecule; and c represents theconcentration of the analyte molecule, provided that when x is 0, c isc_(s); andθ=R/R max   (3)wherein θ represents an adsorption ratio (=amount of adsorption/amountof saturated adsorption); R represents a surface plasmon signal; andRmax represents a signal resulting when an analyte molecule issaturatedly adsorbed.

Preferably, the dissociation constant is determined while makingcorrections based on the supposition that the time required for theliquid exchange is an adsorption phenomenon under ideal conditions.

Preferably, the dissociation constant is determined by using nonlinearregression analysis.

Preferably, a surface plasmon resonance measurement device is used,which comprises a dielectric block, a metal film formed on one side ofthe dielectric block, a light source for generating a light beam, anoptical system for allowing the above light beam to enter the abovedielectric block so that total reflection conditions can be obtained atthe interface between the dielectric block and the metal film and sothat various incidence angles can be included, a flow channel systemcomprising a cell formed on the above metal film, and a light-detectingmeans for detecting the state of surface plasmon resonance by measuringthe intensity of the light beam totally reflected at the aboveinterface.

Preferably, the liquid contained in the above flow channel system isexchanged from a reference liquid containing no test substance to bemeasured to a sample liquid containing a test substance to be measured,and thereafter, a change in surface plasmon resonance is measured in astate where the flow of the sample liquid has been stopped.

Preferably, a reference cell, to which a physiologically activesubstance interacting with a test substance does not bind, is connectedin series with a detection cell, to which a physiologically activesubstance interacting with a test substance binds, the connected cellsare placed in a flow channel system, and a liquid is then fed throughthe reference cell and the detection cell.

Preferably, the ratio (Ve/Vs) of the amount of a liquid exchanged (Veml) in a single measurement to the volume of the above cell (Vs ml) isbetween 1 and 100. More preferably, the ratio (Ve/Vs) is between 1 and50.

Preferably, the time required for the exchange of the liquid containedin the above flow channel system is between 0.01 second and 100 seconds.

BEST MODES FOR CARRYING OUT THE INVENTION

Hereafter, embodiments of the invention will be described.

The present invention relates to a method for measuring dissociationconstants of an analyte molecule immobilized on a metal surface and amolecule that interacts therewith. In the method for measuringdissociation constants according to the present invention, morespecifically, changes in surface plasmon resonance signals are measuredby using a surface plasmon resonance measurement device comprising aflow channel system having a cell formed on a metal film and alight-detecting means for detecting the state of surface plasmonresonance by measuring the intensity of a light beam totally reflectedon the metal film, wherein a change in surface plasmon resonance signalsis measured in a state where the flow of the liquid has been stopped,after the liquid contained in the above flow channel system has beenexchanged, and wherein the dissociation constant is determined based onthe results of measurement of signal changes.

Another aspect of the present invention provides a method for measuringchanges in surface plasmon resonance signals which comprises determiningthe dissociation constant between an analyte molecule immobilized on ametal surface and a molecule that interacts therewith based on theresults of measurement of changes of surface plasmon resonance signalsby using a surface plasmon resonance measurement device comprising aflow channel system having a cell formed on a metal film and alight-detecting means for detecting the state of surface plasmonresonance by measuring the intensity of a light beam totally reflectedon the metal film, wherein a change in surface plasmon resonance signalsis measured in a state where the flow of the liquid has been stopped,after the liquid contained in the above flow channel system has beenexchanged.

The analyte molecule adsorbs to a molecule that interacts therewithimmobilized on the surface of the metal with the elapse of time. Thisphenomenon can be represented by the following equation (1):dθ/dt=ka×c _(s)×(1−θ)−kd×θ  (1)wherein θ represents an adsorption ratio (=amount of adsorption/amountof saturated adsorption); ka represents an adsorption rate coefficient;kd represents a dissociation rate coefficient; and c_(s) represents theconcentration of the analyte molecule that exists near the metalsurface.

Under ideal conditions where a liquid on a metal surface can beconstantly replaced with a fresh liquid, c_(s) is maintained at aconstant level, and ka and kd can be determined based on the assayresults by solving simple difference equations.

However, a liquid flow on the metal surface is very slow, and a solutioncontaining analyte molecules must be flowed rapidly in order to maintainc_(s) at a constant level. In contrast, surface plasmon signal isfluctuated by a disturbed liquid flow on the metal surface, and a largequantity of analyte molecules must be used in order to flow the solutionrapidly. Accordingly, it is practically impossible to maintain c_(s) ata constant level.

When c_(s) is not maintained at a constant level, changes in theconcentration resulting from adsorption/dissociation of analytemolecules are functions that are varied by offshore diffusion of analytemolecules. Such diffusion is represented by the following equation (2):∂c/∂t=D×∂ ² c/∂ ²   (2)wherein x represents a distance from a metal surface; D represents adiffusion coefficient of the analyte molecule; and c represents theconcentration of the analyte molecule, provided that when x is 0, c isc_(s).

The surface plasmon signal R (a difference from the surface plasmonsignal when analyte molecules are not adsorbed) is known to beproportional to the amount of analyte molecules adsorbed on the metalsurface and is represented by the following equation (3):θ=R/R max   (3)wherein θ represents an adsorption ratio (=amount of adsorption/amountof saturated adsorption); R represents a surface plasmon signal; andRmax represents a signal resulting when an analyte molecule issaturatedly adsorbed.

By using the above equations (1), (2), and (3), the adsorption ratecoefficient ka, the dissociation rate coefficient kd, and a ratiothereof, i.e., the equilibrium dissociation coefficient KD (=ka/kd), canbe determined from the signal measured by the experiment.

Hereafter, a method of analysis is described in detail.

When the rate of replacing a reference solution that contains no analytemolecules with a sample solution that contains analyte molecules issufficiently faster than the adsorption rate of analyte molecules andwhen a regular offshore flow of a solution with a concentration of C₀occurs constantly (hereafter such situation is referred to as the “idealcondition”), values of interest can be obtained by solving the equation(1). More specifically, changes in surface plasmon signals arerepresented by equation (4):dR/dt=ka×C0×(R max−R) kd×R   (4).

Changes in the signal R at the time point “t” obtained via experimentare subjected to nonlinear regression analysis using the equation (4).Thus, the adsorption rate coefficient ka and the dissociation ratecoefficient kd can be determined. Since the Rmax value is affected bythe state of analyte molecules bound to a metal surface or otherconditions, it is preferable to determine ka and kd simultaneously. Thisrequires nonlinear regression analysis with 3 unknown quantities. Inorder to fulfill such requirement, however, the linear velocity of asolution must be increased. This disadvantageously results in anunstabilized surface plasmon signal and an increased noise level.

When the rate of replacing a reference sample that contains no analytemolecules with a sample solution that contains analyte molecules atconcentration of C₀ is sufficiently faster than the adsorption rate ofanalyte molecules and the liquid flow is stopped immediately afterliquid exchange (hereafter this is referred to as the “stopped flow (SF)conditions), the adsorption rate coefficient ka and the dissociationrate coefficient kd can be determined using the equation (1), (2), or(3). Specifically, in the equations (1), (2), and (3), differenceequations can be approximately replaced with the following differenceequations.R[t]=(ka×C[0,(t−Δt)]×(R max−R[t−Δt])−kd×R[t−Δt])×Δt+R[t−Δt)   (5)C[0,t]=D×(C[Δx,(t−Δt)]−C[0,(t−Δt)])+C[0,(t−Δt)]−(ka×C[0,(t−Δt)]×(Rmax−R[t−Δt])−(kd×R[t−Δt])/R max×Sm×Δt)/Δx   (6)C[n×Δx,t]=D×(C[(n+1)×Δx, (t−Δt)]+C[(n−1)×Δx,(t−Δt)]−2×C[Δx,(t−Δt)])+C[Δx, (t−Δt)]  (7)wherein R[t] represents a surface plasmon signal at the time point t; Δtrepresents a minute time; C[x, t] represents a concentration of theanalyte molecule at a point that is the distance x away from a metalsurface at the time point t; Δx represents a minute distance; and Smrepresents the amount of saturatedly adsorbed analyte molecules.

When Δx and Δt are sufficiently small, the equations (5), (6), and (7)are approximated by the equations (1), (2), and (3). In this case,diffusion of analyte molecules at the time point Δt must be sufficientlyshorter than the distance Δx. More specifically, the tentative diffusioncoefficient D_(M) represented by the following equation (8) must bebetween 0.01 and 0.5, and it is preferably between 0.05 and 0.45.Δx ² /D×Δt=D _(M)   (8)

Further, the level of changes in the concentration of analyte moleculesat a point of contact with a metal surface should be sufficiently smallat the time point Δt. It is represented by the equation (9):(C[0,t+Δt]−C[0,t])<<C[0,t]  (9)

It is necessary for Δx to fulfill the following equation (10) fromequations (6), (8), or (9):Δx <<D/(D _(M) ×Sm×ka)   (10)

When the equation (10) is expressed as the following equation (11) usingthe degree of incidence constant Ax, Ax must be between 0.0001 and 0.1,and it is preferably between 0.001 and 0.03.Δx=Ax×D/(D _(M) ×Sm×ka)   (11)

When usual assay results are analyzed, calculation is performed while karemains unknown. Accordingly, Δx must be determined by assigning a valuethat is larger than the maximum value, which is deduced to be ka value.

The surface plasmon signal R[t] can be represented by applying theboundary conditions of the following equation (12) to the aforementionedequations (4) to (11).C[n×Δx,0]=C ₀   (12)

Under the SF conditions, the rate of replacing a reference solution thatcontains no analyte molecules with a sample solution that containsanalyte molecules at a concentration C₀ is deduced to be sufficientlyfaster than the adsorption rate of analyte molecules. Such deduction isdifficult to prove in an actual assay system (under real SF conditions).Accordingly, changes occur more rapidly than SF conditions upon liquidexchange, as shown in FIG. 3. Since liquid flows at a high speed at thetime of liquid exchange, assay cannot be sufficiently carried out due tonoises. However, adsorption is observed under ideal conditions.

In actual analysis, the following difference equation (13) is calculatedbased on the equation (4) during the time of liquid exchange(0≦t≦t_(i)). Analysis can be carried out in accordance with the equation(14), (15), or (16) after the liquid exchange (t_(i)≦t). Values thatsatisfy the requirements of equations (8) to (11) are selected for Δxand Δt.

When the condition 0≦t≦t_(i) is satisfied:R[t]=(ka×C ₀×(R max−R[t−Δt])−kd×R[t−Δt])×Δt+R[t−Δt]  (13).

When the condition t_(i)≦t is satisfied: $\begin{matrix}\begin{matrix}{{R\lbrack t\rbrack} = \left( {{{ka} \times {C\left\lbrack {0,\left( {t - {\Delta\quad t}} \right)} \right\rbrack} \times \left( {{Rmax} - {R\left\lbrack {t - {\Delta\quad t}} \right\rbrack}} \right)} -} \right.} \\{{{\left. {{kd} \times {R\left\lbrack {t - {\Delta\quad t}} \right\rbrack}} \right) \times \Delta\quad t} + {R\left\lbrack {t - {\Delta\quad t}} \right\rbrack}},}\end{matrix} & (14) \\\begin{matrix}{{C\left\lbrack {0,t} \right\rbrack} = {{D \times \left( {{C\left\lbrack {{\Delta\quad x},\left( {t - {\Delta\quad t}} \right)} \right\rbrack} - {C\left\lbrack {0,\left( {t - {\Delta\quad t}} \right)} \right\rbrack}} \right)} +}} \\{{C\left\lbrack {0,\left( {t - {\Delta\quad t}} \right)} \right\rbrack} - \left( {{ka} \times {C\left\lbrack {0,\left( {t - {\Delta\quad t}} \right)} \right\rbrack} \times} \right.} \\{\left( {{Rmax} - {R\left\lbrack {t - {\Delta\quad t}} \right\rbrack}} \right) - {\left( {{kd} \times {R\left\lbrack {t - {\Delta\quad t}} \right\rbrack}} \right)/}} \\{{\left. {{Rmax} \times {Sm} \times \Delta\quad t} \right)/\Delta}\quad x}\end{matrix} & (15) \\\begin{matrix}{{C\left\lbrack {{n \times \Delta\quad x},t} \right\rbrack} = {D \times \left( {{C\left\lbrack {{\left( {n + 1} \right) \times \Delta\quad x},\left( {t - {\Delta\quad t}} \right)} \right\rbrack} +} \right.}} \\{{C\left\lbrack {{\left( {n - 1} \right) \times \Delta\quad x},\left( {t - {\Delta\quad t}} \right)} \right\rbrack} - {2 \times}} \\{\left. {C\left\lbrack {{\Delta\quad x},\left( {t - {\Delta\quad t}} \right)} \right\rbrack} \right) + {C\left\lbrack {{\Delta\quad x},\left( {t - {\Delta\quad t}} \right)} \right\rbrack}}\end{matrix} & (16)\end{matrix}$

The equation (17) is employed as the boundary condition.C[n×Δx,t _(i) ]=C ₀   (17)

In order to subject the actual assay results to nonlinear regressionanalysis under the real SF conditions, it is preferable to handle thediffusion coefficient D as an unknown quantity in addition to ka, kd,and Rmax. The diffusion coefficient D is considered to be determinedfrom a molecular weight by using the Einstein's equation. In practice,influences of molecular configurations or solvents are significant, andthus, it is preferable to actually perform measurement. In the 0≦t≦t_(i)region, noises generated in the assay system is large, and thus, usethereof for calculation results in a larger error. Thus, nonlinearregression analysis is carried out concerning the t_(i)≦t region byemploying ka, kd, Rmax, and D as unknown quantities. The curveindicating the entire 0≦t region obtained from the obtained ka, kd,Rmax, and D is compared with the curve indicating the measured value.These curves are adequately consistent with each other, as shown in FIG.4.

Actual calculation may be carried out via any type of algorithm as longas any of the difference equations (13) to (17) is employed. Also, nonregression analysis may be carried out via any conventional techniques.

In the present invention, the noise width of a change in signals of areference cell during measurement and base line fluctuation can besuppressed by measuring a change in surface plasmon resonance in a statewhere the flow of a liquid has been stopped, so that binding detectiondata with high reliability can be obtained. The time of the stop of theflow of the liquid is not particularly limited. For example, it may bebetween 1 second and 30 minutes, preferably between 10 seconds and 20minutes, and more preferably between 1 minute and 20 minutes.

In the present invention, preferably, the liquid contained in a flowchannel system is exchanged from a reference liquid containing no testsubstance to be measured to a sample liquid containing a test substanceto be measured, and thereafter, a change in surface plasmon resonancecan be measured in a state where the flow of the sample liquid has beenstopped.

In the present invention, preferably, a reference cell, to which asubstance interacting with a test substance does not bind, is connectedin series with a detection cell, to which a substance interacting with atest substance binds, the connected cells are placed in a flow channelsystem, and a liquid is then fed through the reference cell and thedetection cell, so that a change in surface plasmon resonance can bemeasured.

In addition, in the present invention, the ratio (Ve/Vs) of the amountof a liquid exchanged (Ve ml) in a single measurement to the volume (Vsml) of a cell used in measurement (and when the aforementioned referencecell and detection cell are used, the total volume of these cells) ispreferably between 1 and 100. Ve/Vs is more preferably between 1 and 50,and particularly preferably between 1 and 20. The volume (Vs ml) of acell used in measurement is not particularly limited. It is preferablybetween 1×10⁻⁶ and 1.0 ml, and particularly preferably between 1×10⁻⁵and 1×10⁻¹ ml. The period of time necessary for exchanging the liquid ispreferably between 0.01 second and 100 seconds, and particularlypreferably between 0.1 second and 10 seconds.

The surface plasmon resonance phenomenon occurs due to the fact that theintensity of monochromatic light reflected from the border between anoptically transparent substance such as glass and a metal thin filmlayer depends on the refractive index of a sample located on theoutgoing side of the metal. Accordingly, the sample can be analyzed bymeasuring the intensity of reflected monochromatic light. The surfaceplasmon resonance measurement device used in the present invention willbe described below.

The surface plasmon resonance measurement device is a device foranalyzing the properties of a substance to be measured using aphenomenon whereby a surface plasmon is excited with a lightwave. Thesurface plasmon resonance measurement device used in the presentinvention comprises a dielectric block, a metal film formed on a face ofthe dielectric block, a light source for generating a light beam, anoptical system for allowing the above light beam to enter the abovedielectric block such that total reflection conditions can be obtainedat the interface between the above dielectric block and the above metalfilm and that components at various incident angles can be contained,and a light-detecting means for detecting the state of surface plasmonresonance by measuring the intensity of the light beam totally reflectedat the above interface.

Moreover, as stated above, the above dielectric block is formed as oneblock comprising the entity of the entrance face and exit face of theabove light beam and a face on which the above metal film is formed, andthe above metal film is integrated with this dielectric block.

In the present invention, more specifically, a surface plasmon resonancemeasurement device shown in FIGS. 1 to 32 of Japanese Patent Laid-OpenNo. 2001-330560, and a surface plasmon resonance device shown in FIGS. 1to 15 of Japanese Patent Laid-Open No. 2002-296177, can be preferablyused. All of the contents as disclosed in Japanese Patent Laid-Open Nos.2001-330560 and 2002-296177 cited in the present specification areincorporated herein by reference as a part of the disclosure of thisspecification.

For example, the surface plasmon resonance measurement device describedin Japanese Patent Laid-Open No. 2001-330560 is characterized in that itcomprises: a dielectric block; a thin metal film formed on a face of thedielectric block; multiple measurement units comprising asample-retaining mechanism for retaining a sample on the surface of thethin film; a supporting medium for supporting the multiple measurementunits; a light source for generating a light beam; an optical system forallowing the above light beam to enter the dielectric block at variousangles so that total reflection conditions can be obtained at theinterface between the dielectric block and the metal film; alight-detecting means for measuring the intensity of the light beamtotally reflected at the above interface and detecting the state ofattenuated total reflection caused by surface plasmon resonance; and adriving means for making the above supporting medium, the above opticalsystem and the above light-detecting means move relative to one another,and successively placing each of the above multiple measurement units ina certain position appropriate to the above optical system and the abovelight-detecting means, so that the above total reflection conditions andvarious incident angles can be obtained with respect to each dielectricblock of the above multiple measurement units.

It is to be noted that in the above measurement device, the aboveoptical system and light-detecting means are kept in a resting state andthe above driving means makes the above supporting medium move.

In such a case, the above supporting medium is desirably a turntable forsupporting the above multiple measurement units on a circle centered ona rotation axis, and the above driving means is desirably a means forintermittently rotating this turntable. In this case, a medium forsupporting the above multiple measurement units that are linearlyarranged in a line may be used as the above supporting medium, and ameans that makes such a supporting medium move linearly in anintermittent fashion in the direction in which the above multiplemeasurement units are arranged may be applied as the above drivingmeans.

Otherwise, on the contrary, it may also be possible that the abovesupporting medium be retained in a resting state and that the abovedriving means makes the above optical system and light-detecting meansmove.

In such a case, the above supporting medium is desirably a medium forsupporting the above multiple measurement units on a circle, and theabove driving means is desirably a means for intermittently rotating theabove optical system and light-detecting means along the multiplemeasurement units supported by the above supporting medium. In thiscase, a medium for supporting the above multiple measurement units thatare linearly arranged in a line may be used as the above supportingmedium, and a means that makes the above optical system andlight-detecting means move linearly in an intermittent fashion along themultiple measurement units supported by the above supporting medium maybe applied as the above driving means.

Otherwise, when the above driving means has a rolling bearing thatsupports a rotation axis, the driving means is desirably configured suchthat after the rotation axis has been rotated to a certain direction anda series of measurements for the above multiple measurement units hasbeen terminated, the above rotation axis is equivalently rotated to theopposite direction, and then it is rotated again to the same abovedirection for the next series of measurements.

In addition, the above-described measurement device is desirablyconfigured such that the above multiple measurement units are connectedin a line with a connecting member so as to constitute a unit connectedbody and that the above supporting medium supports the unit connectedbody.

Moreover, in the above-described measurement device, it is desirable toestablish a means for automatically feeding a given sample to eachsample-retaining mechanism of the multiple measurement units supportedby the above supporting medium.

Furthermore, in the above-described measurement device, it is desirablethat the dielectric block of the above measurement unit be immobilizedto the above supporting medium, that a thin film layer and asample-retaining mechanism of the measurement unit be unified so as toconstitute a measurement chip, and that the measurement chip be formedsuch that it is exchangeable with respect to the above dielectric block.

When such a measurement chip is applied, it is desirable to establish acassette for accommodating a multiple number of the measurement chipsand a chip-supplying means for successively taking a measurement chipout of the cassette and supplying it in a state in which it is connectedto the above dielectric block.

Otherwise, it may also be possible to unify the dielectric block of themeasurement unit, the thin film layer and the sample-retainingmechanism, so as to constitute a measurement chip, and it may also bepossible for this measurement chip to be formed such that it isexchangeable with respect to the above supporting medium.

When a measurement chip has such a structure, it is desirable toestablish a cassette for accommodating a multiple number of measurementchips and a chip-supplying means for successively taking a measurementchip out of the cassette and supplying it in a state in which it issupported by the supporting medium.

The above optical system is desirably configured such that it makes alight beam enter the dielectric block in a state of convergent light ordivergent light. Moreover, the above light-detecting means is desirablyconfigured such that it detects the position of a dark line generateddue to attenuated total reflection, which exists in the totallyreflected light beam.

Furthermore, the above optical system is desirably configured such thatit makes a light beam enter the above interface in a defocused state. Inthis case, the beam diameter of the light beam at the above interface ina direction wherein the above supporting medium moves is desirably tentimes or greater the mechanical positioning precision of the abovesupporting medium.

Still further, the above-described measurement device is desirablyconfigured such that the measurement unit is supported on the upper sideof the above supporting medium, such that the above light source isplaced so as to project the above light beam from a position above theabove supporting medium to downwards, and such that the above opticalsystem comprises a reflecting member for reflecting upwards the abovelight beam projected to downwards as described above and making itproceed towards the above interface.

Still further, the above-described measurement device is desirablyconfigured such that the above measurement unit is supported on theupper side of the above supporting medium, such that the above opticalsystem is constituted so as to make the above light beam enter the aboveinterface from the downside thereof, and such that the abovelight-detecting means is placed in a position above the above supportingmedium with a light-detecting plane thereof facing downwards, as well ascomprising a reflecting member for reflecting upwards the totallyreflected light beam at the above interface and making it proceedtowards the above light-detecting means.

What is more, the above-described measurement device desirably comprisesa temperature-controlling means for maintaining the temperature of theabove measurement unit before and/or after being supported by the abovesupporting medium at a predetermined temperature.

Moreover, the above-described measurement device desirably comprises ameans for stirring the sample stored in the sample-retaining mechanismof the measurement unit supported by the above supporting medium beforedetecting the state of attenuated total reflection as mentioned above.

Furthermore, in the above-described measurement device, it is desirableto establish in at least one of the multiple measurement units supportedby the above supporting medium a standard solution-supplying means forsupplying a standard solution having optical properties associated withthe optical properties of the above sample, as well as a correctingmeans for correcting data regarding the above attenuated totalreflection state of the sample based on the data regarding the aboveattenuated total reflection state of the above standard solution.

In such a case, if the sample is obtained by dissolving a test substancein a solvent, it is desirable that the above standard solution-supplyingmeans be a means for supplying the above solvent as a standard solution.

Still further, the above measurement device desirably comprises: a markfor indicating individual recognition information; a reading means forreading the above mark from the measurement unit used in measurement; aninputting means for inputting sample information regarding the samplesupplied to the measurement unit; a displaying means for displayingmeasurement results; and a controlling means connected to the abovedisplaying means, inputting means and reading means, which stores theabove individual recognition information and sample information of eachmeasurement unit while associating them with each other, as well asmaking the above displaying means display the measurement results of thesample retained in a certain measurement unit while associating themwith the above individual recognition information and sample informationof each measurement unit.

When a substance interacting with a physiologically active substance isdetected or measured using the above-described measurement device, astate of attenuated total reflection is detected in a sample containedin one of the above measurement units, and thereafter, the abovesupporting medium, optical system and light-detecting means are movedrelative to one another, so that a state of attenuated total reflectionis detected in a sample contained in another measurement unit.Thereafter, the above supporting medium, optical system andlight-detecting means are again moved relative to one another, so that astate of attenuated total reflection is detected again the samplecontained in the above one measurement unit, thereby completing themeasurement.

The measurement chip used in the present invention is used for thesurface plasmon resonance measurement device having a structuredescribed herein, and comprises a dielectric block and a metal filmformed on a face of the dielectric block, in which the dielectric blockis formed as one block comprising the entirety of the entrance face andexit face of the light beam and a face on which the above metal film isformed, the above metal film is integrated with the above dielectricblock.

A metal constituting the metal film is not particularly limited, as longas surface plasmon resonance is generated. Examples of a preferred metalmay include free-electron metals such as gold, silver, copper, aluminumor platinum. Of these, gold is particularly preferable. These metals canbe used singly or in combination. Moreover, considering adherability tothe above substrate, an interstitial layer consisting of chrome or thelike may be provided between the substrate and a metal layer.

The film thickness of a metal film is not limited. When the metal filmis used for a surface plasmon resonance biosensor, the thickness ispreferably between 0.1 nm and 500 nm, and particularly preferablybetween 1 nm and 200 nm. If the thickness exceeds 500 nm, the surfaceplasmon phenomenon of a medium cannot be sufficiently detected.Moreover, when an interstitial layer consisting of chrome or the like isprovided, the thickness of the interstitial layer is preferably between0.1 nm and 10 nm.

Formation of a metal film may be carried out by common methods, andexamples of such a method may include sputtering method, evaporationmethod, ion plating method, electroplating method, and nonelectrolyticplating method.

A metal film is preferably placed on a substrate. The description“placed on a substrate” is used herein to mean a case where a metal filmis placed on a substrate such that it directly comes into contact withthe substrate, as well as a case where a metal film is placed viaanother layer without directly coming into contact with the substrate.When a substrate used in the present invention is used for a surfaceplasmon resonance biosensor, examples of such a substrate may include,generally, optical glasses such as BK7, and synthetic resins. Morespecifically, materials transparent to laser beams, such as polymethylmethacrylate, polyethylene terephthalate, polycarbonate or a cycloolefinpolymer, can be used. For such a substrate, materials that are notanisotropic with regard to polarized light and having excellentworkability are preferably used.

Preferably, the metal film has a functional group capable ofimmobilizing a physiologically active substance on the outermost surfaceof the substrate. The term “the outermost surface of the substrate” isused to mean “the surface, which is farthest from the substrate”.

Examples of a preferred functional group may include —OH, —SH, —COOH,—NR¹R² (wherein each of R¹ and R² independently represents a hydrogenatom or lower alkyl group), —CHO, —NR³NR¹R² (wherein each of R¹, R²andR³ independently represents a hydrogen atom or lower alkyl group), —NCO,—NCS, an epoxy group, and a vinyl group. The number of carbon atomscontained in the lower alkyl group is not particularly limited herein.However, it is generally about C1 to C10, and preferably C1 to C6.

Examples of the method of introducing such a functional group include amethod which involves applying a polymer containing a precursor of sucha functional group on a metal surface or metal film, and then generatingthe functional group from the precursor located on the outermost surfaceby chemical treatment.

In the measurement chip obtained as mentioned above, a physiologicallyactive substance is covalently bound thereto via the above functionalgroup, so that the physiologically active substance can be immobilizedon the metal film.

A physiologically active substance immobilized on the surface for themeasurment chip of the present invention is not particularly limited, aslong as it interacts with a measurement target. Examples of such asubstance may include an immune protein, an enzyme, a microorganism,nucleic acid, a low molecular weight organic compound, a nonimmuneprotein, an immunoglobulin-binding protein, a sugar-binding protein, asugar chain recognizing sugar, fatty acid or fatty acid ester, andpolypeptide or oligopeptide having a ligand-binding ability.

Examples of an immune protein may include an antibody whose antigen is ameasurement target, and a hapten. Examples of such an antibody mayinclude various immunoglobulins such as IgG, IgM, IgA, IgE or IgD. Morespecifically, when a measurement target is human serum albumin, ananti-human serum albumin antibody can be used as an antibody. When anantigen is an agricultural chemical, pesticide, methicillin-resistantStaphylococcus aureus, antibiotic, narcotic drug, cocaine, heroin, crackor the like, there can be used, for example, an anti-atrazine antibody,anti-kanamycin antibody, anti-metamphetamine antibody, or antibodiesagainst O antigens 26, 86, 55, 111 and 157 among enteropathogenicEscherichia coli.

An enzyme used as a physiologically active substance herein is notparticularly limited, as long as it exhibits an activity to ameasurement target or substance metabolized from the measurement target.Various enzymes such as oxidoreductase, hydrolase, isomerase, lyase orsynthetase can be used. More specifically, when a measurement target isglucose, glucose oxidase is used, and when a measurement target ischolesterol, cholesterol oxidase is used. Moreover, when a measurementtarget is an agricultural chemical, pesticide, methicillin-resistantStaphylococcus aureus, antibiotic, narcotic drug, cocaine, heroin, crackor the like, enzymes such as acetylcholine esterase, catecholamineesterase, noradrenalin esterase or dopamine esterase, which show aspecific reaction with a substance metabolized from the abovemeasurement target, can be used.

A microorganism used as a physiologically active substance herein is notparticularly limited, and various microorganisms such as Escherichiacoil can be used.

As nucleic acid, those complementarily hybridizing with nucleic acid asa measurement target can be used. Either DNA (including cDNA) or RNA canbe used as nucleic acid. The type of DNA is not particularly limited,and any of native DNA, recombinant DNA produced by gene recombinationand chemically synthesized DNA may be used.

As a low molecular weight organic compound, any given compound that canbe synthesized by a common method of synthesizing an organic compoundcan be used.

A nonimmune protein used herein is not particularly limited, andexamples of such a nonimmune protein may include avidin (streptoavidin),biotin, and a receptor.

Examples of an immunoglobulin-binding protein used herein may includeprotein A, protein G, and a rheumatoid factor (RF).

As a sugar-binding protein, for example, lectin is used.

Examples of fatty acid or fatty acid ester may include stearic acid,arachidic acid, behenic acid, ethyl stearate, ethyl arachidate, andethyl behenate.

When a physiologically active substance is a protein such as an antibodyor enzyme, or nucleic acid, an amino group, thiol group or the like ofthe physiologically active substance is covalently bound to a functionalgroup located on a metal surface, so that the physiologically activesubstance can be immobilized on the metal surface.

A measurement chip to which a physiologically active substance isimmobilized as described above can be used to detect and/or measure asubstance which interacts with the physiologically active substance.

Namely, the present invention provides a method for detecting ormeasuring a substance interacting with a physiologically activesubstance, which comprises steps of: using at least a measurement chip(cell), to the surface of which a physiologically active substance bindsby covalent bonding; allowing a sample liquid containing a testsubstance to be measured to come into contact with the above cell; andafter exchanging the liquid contained in a flow channel system,measuring a change in surface plasmon resonance in a state where theflow of the liquid has been stopped.

As a test substance, a sample containing a substance interacting withthe aforementioned physiologically active substance can be used, forexample.

The present invention will be described in detail by the followingexamples, but the present invention is not limited thereto.

EXAMPLES

The following experiment was carried out using a device shown in FIG. 22of Japanese Patent Laid-Open No. 2001-330560 (hereinafter referred to asthe surface plasmon resonance measurement device of the presentinvention) (shown in FIG. 1 of the present specification) and adielectric block shown in FIG. 23 of Japanese Patent Laid-Open No.2001-330560 (hereinafter referred to as the dielectric block of thepresent invention) (shown in FIG. 2 of the present specification).

In the surface plasmon resonance measurement device shown in FIG. 1, aslide block 401 is used as a supporting medium for supportingmeasurement units, which is joined to two guide rods 400, 400 placed inparallel with each other while flexibly sliding in contact, and whichalso flexibly moves linearly along the two rods in the direction of anarrow Y in the figure. The slide block 401 is screwed together with aprecision screw 402 placed in parallel with the above guide rods 400,400, and the precision screw 402 is reciprocally rotated by a pulsemotor 403 which constitutes a supporting medium-driving means togetherwith the precision screw 402.

It is to be noted that the movement of the pulse motor 403 is controlledby a motor controller 404. This is to say, an output signal S 40 of alinear encoder (not shown in the figure), which is incorporated into theslide block 401 and detects the position of the slide block 401 in thelongitudinal direction of the guide rods 400, 400, is inputted into themotor controller 404. The motor controller 404 controls the movement ofthe pulse motor 403 based on the signal S 40.

Moreover, below the guide rods 400, 400, there are established a laserlight source 31 and a condenser 32 such that they sandwich from bothsides the slide block 401 moving along the guide rods, and aphotodetector 40. The condenser 32 condenses a light beam 30. Inaddition, the photodetector 40 is placed thereon.

In this embodiment, a stick-form unit connected body 410 obtained byconnecting and fixing eight measurement units 10 is used as an example,and the measurement units 10 are mounted on the slide block 401 in astate in which eight units are arranged in a line.

FIG. 2 shows the structure of the unit connected body 410 in detail. Asshown in the figure, the unit connected body 410 is obtained byconnecting the eight measurement units 10 by a connecting member 411.

This measurement unit 10 is obtained by molding a dielectric block 11and a sample-retaining frame 13 into one piece, for example, usingtransparent resin or the like. The measurement unit constitutes ameasurement chip that is exchangeable with respect to a turntable. Inorder to make the measurement chip exchangeable, for example, themeasurement unit 10 may be fitted into a through-hole that is formed inthe turntable. In the present example, a sensing substance 14 isimmobilized on a metal film 12.

Examples: Measurement in a State where the Flow of the Liquid has beenStopped

(1) Preparation of Dextran Assay Chip

The dielectric block of the present invention onto which gold having athickness of 50 nm had been evaporated as a metal film was treated witha Model-208 UV ozone cleaning system (TECHNOVISION INC.) for 30 minutes.Then, 5.0 mM solution of 11-hydroxy-1-undecanethiol in ethanol/water(80/20) was allowed to come into contact with the metal film, andsurface treatment was carried out at 25° C. for 18 hours. Thereafter,the resultant product was washed with ethanol 5 times, with a mixedsolvent consisting of ethanol and water 1 time, and then with water 5times.

Subsequently, the surface coated with 11-hydroxy-1-undecanethiol wasallowed to come into contact with a 10% by weight of epichlorohydrinsolution (a solvent: a mixed solution of 0.4M sodium hydroxide anddiethylene glycol dimethyl ether at a ratio of 1:1), and the reactionwas allowed to proceed in a shaking incubator at 25° C. for 4 hours. Thesurface was washed twice with ethanol and 5 times with water.

Subsequently, 4.5 ml of 1M sodium hydroxide was added to 40.5 ml of 25%by weight of dextran (T500, Pharmacia) aqueous solution. The resultingsolution was allowed to come into contact with the surface that had beentreated with epichlorohydrin. The surface was then incubated in ashaking incubator at 25° C. for 20 hours. The surface was washed withwater 10 times at 50° C. Subsequently, 3.5 g of bromoacetic acid wasdissolved in 27 g of a 2M sodium hydroxide solution, the resultingmixture was allowed to come into contact with the dextran-treatedsurface, and the surface was then incubated in a shaking incubator at28° C. for 16 hours. The surface was washed with water, and theprocedure described above was repeated once.

(2) Preparation of a Chip having Protein A Immobilized Thereon:

After a solution in the dextran measurement chip prepared in the above(1) was removed, 70 μl of a mixed solution of 200 mM EDC(N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride) and 50 mMNHS (N-hydroxysuccinimide) was added thereto, and the resultant was thenallowed to stand for 10 minutes. After the mixed solution was removed,the chip was washed three times with 100 μl of water and then threetimes with 100 μl of acetate 5.0 buffer (BIAcore). The chip was mountedon the surface plasmon resonance measurement device of the presentinvention while containing 100 μl of acetate 5.0 buffer, the inside ofthe chip was replaced with a protein A solution (a solution of 50 μg/mlprotein A (Nacalai Tesque Inc.) in acetate 5.0 buffer (BIAcore)), andthe chip was allowed to stand for 30 minutes so that protein A wasimmobilized thereon. The inside of the chip was replaced with 1Methanolamine solution and the chip was allowed to stand for 10 minutes.The inside of the chip was washed ten times with 100 μl of acetate 5.0buffer. Change in the resonance signal resulting from protein Aimmobilization was 500 RU.

(3) Preparation of a Reference Chip:

After a solution in the dextran measurement chip prepared in (1) abovewas removed, 70 μl of a mixed solution of 200 mM EDC and 50 mM NHS wasadded thereto, and the resultant was then allowed to stand for 10minutes. After the mixed solution was removed, the chip was washed threetimes with 100 μl of water and then three times with 100 μl of acetate5.0 buffer. The inside of the chip was replaced with 1M ethanolaminesolution and the chip was allowed to stand for 10 minutes. The inside ofthe chip was washed ten times with 100 μl of acetate 5.0 buffer.

(4) Preparation of a Flow Channel System:

A dielectric block was mounted on the chip having protein A immobilizedthereon according to the present invention, and sealed with siliconrubber to prepare a cell having an inner volume of 15 μl. Two holes of adiameter of 1 mm were provided on the silicon rubber seal, and these twoholes were connected with each other via a tefron tube having an innerdiameter of 0.5 mm and an outer diameter of 1 mm to prepare a flowchannel. Similarly, a cover and a flow channel were provided for thereference chip, these two chips were connected in series with eachother, and the flow channel system was prepared. These two chipscomprising the flow channel were mounted on the surface plasmonresonance measurement device of the present invention.

(5) Evaluation of Binding Capacity for Mouse IgG:

The flow channel system was filled with HBS-EP buffer (BIAcore). HBS-EPbuffer was composed of 0.01 mol/l of HEPES(N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid) (pH 7.4), 0.15mol/l of NaCl, 0.003 mol/l of EDTA, and 0.005% by weight of SurfactantP20. Changes in the signal levels were assayed at intervals of 0.5seconds by employing the signal level before liquid exchange as thebaseline. The inside of the flow channel system was replaced with amouse IgG solution (a solution of 10 μg/ml mouse IgG (purchased fromCosmo Bio Co., Ltd) in HBS-EP buffer) at 20 μl/sec. Such replacement wascompleted in 5 seconds.

The liquid flow was stopped 5 seconds after the initation of liquidexchange.

The data acquired 5 seconds to 2 minutes after the initation of liquidexchange were used, initial values, ka=1×10⁵ (M⁻¹s⁻¹), kd=1×10⁻³ (s⁻¹),Rmax=500 (RU), D=1×10⁻⁷ (cm² s⁻¹), and C=6.7×10⁻⁸ (M), were assigned,and ka, kd, Rmax, and D values were converted using the Solver functions(Excel 2000, Microsoft). The curve exhibiting the smallest errors by themethod of least squares from the curve indicating the measured value wasdetermined by the nonlinear regression method. The results are shown inFIG. 4. This demonstrates that the curve indicating the measured valueis substantially consistent with the curve indicating the calculatedvalue.

Comparative Example: Measurement in a State where Liquid is Flowing

The following experiment was carried out using the BlAcore 3000(BIAcore). The sensorchip CM-5 was mounted by a conventional method, andpriming was performed using HBS-EP buffer. A buffer and varioussolutions were allowed to flow through the flow cell 2 at a flow rate of10 μl/min to perform the experiment. After the mixed solution of 200 mMEDC and 50 mM NHS was allowed to flow for 7 minutes, the HBS-EP bufferwas allowed to flow to wash the cell. Subsequently, a protein A solution(Protein A, Nacalai Tesque, Inc.) was dissolved in Acetate 5.0 (BIAcore)to 10 μg/ml, and the resulting solution was allowed to flow therethroughfor 1 minute so as to immobilize protein A. After the protein A solutionwas washed with the HBS-EP buffer, a 1M ethanolamine solution wasallowed to flow for 7 minutes. The ethanolamine solution was washed withthe HBS-EP buffer. Changes in resonance signals resulting fromimmobilization of protein A were 300 RU.

A buffer and various solutions were allowed to flow through the flowcell 1 at a flow rate of 10 μl/min to perform the experiment. After themixed solution of 200 mM EDC and 50 mM NHS was allowed to flow for 7minutes, the HBS-EP buffer was allowed to flow to wash the cell. A 1Methanolamine solution was allowed to flow for 7 minutes. Theethanolamine solution was washed with the HBS-EP buffer.

A buffer and various solutions were allowed to flow through the flowcells 1 and 2 at a flow rate of 20 μl/min to perform the experiment. Amouse IgG solution (a solution of mouse IgG (purchased from Cosmo Bio)dissolved in HBS-EP buffer to 10 μg/ml) was injected using “kinject”command, and binding and dissociation signals were measured 5 minutes.

The measured signals were subjected to fitting using an analyticalsoftware, BIA evaluation 3.1 (BIAcore), in the 1:1 (Langmuir) bindingmode, so as to determine ka, kd, and Rmax.

Parameters obtained in Examples and Comparative Example are shown inTable 1. ka and kd values obtained in Examples were found to be similarto values obtained in Comparative Example. Also, the dissociationconstant with satisfactory baseline fluctuations could be determined inthe same manner as with the ideal conditions. TABLE 1 Amount of Baselineanalyte Noise fluctuation solution used ka kd Rmax D (RU) (RU/min) (μl)Example 3.5 × 10⁵ 9.0 × 10⁻⁴ 640 4.1 × 10⁻⁸ <1 <1 100 Comparative 2.6 ×10⁵ 7.8 × 10⁻⁴ 425 — 6 5 140 Example

INDUSTRIALLY APPLICABILITY

According to the present invention, the dissociation constant of ananalyte molecule immobilized on a metal surface and a molecule thatinteracts therewith can be determined by surface plasmon resonance (SPR)analysis by a method for measuring changes in surface plasmon resonancesignals in a state where the flow of the liquid has been stopped, afterthe liquid contained in the above flow channel system of the plasmonresonance measurement device has been exchanged, which can yield highlyreliable results of measurement with a low noise level (i.e., a noisewidth of a control chip) and small baseline fluctuations (i.e., signalchanges of a control chip).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a surface plasmon resonance measurement device used in theexamples, wherein 10 indicates measurement unit, 30 indicates a lightbeam, 31 indicates laser light source, 32 indicates condenser lens, 40indicates light detector, S40 indicates output signal, 400 indicatesguide rod, 401 indicates slide block, 402 indicates precision screw, 403indicates pulse motor, 404 indicates motor controller, and 410 indicatesunit connector.

FIG. 2 shows the dielectric block used in the examples, wherein 10indicates measurement unit, 11 indicates dielectric block, 12 indicatesmetal film, 13 indicates sample-retaining frame, 14 indicates sensingsubstance, 410 indicates unit connector, and 411 indicates connectingmember.

FIG. 3 shows a comparison of the SPR curve attained under the idealconditions, that attained under the stop-flow conditions, and themeasured SPR curve.

FIG. 4 shows the results of a comparison of the curves of all theregions obtained from ka, kd, Rmax, and D determined by the method ofthe present invention with the measured SPR curve.

1. A method for determining a dissociation constant of an analyte molecule immobilized on a metal surface and a molecule that interacts therewith, wherein changes in surface plasmon resonance signals are measured by using a surface plasmon resonance measurement device comprising a flow channel system having a cell formed on a metal film and a light-detecting means for detecting the state of surface plasmon resonance by measuring the intensity of a light beam totally reflected on the metal film; wherein a change in surface plasmon resonance signals is measured in a state where the flow of the liquid has been stopped, after the liquid contained in the above flow channel system has been exchanged; and wherein the dissociation constant is determined based on the results of measurement of signal changes.
 2. The method according to claim 1, wherein an adsorption rate coefficient (ka) and a dissociation rate coefficient (kd) are determined based on the results of measurement of changes in surface plasmon resonance signals, and a dissociation constant (KD) is determined using an equation represented by KD=kd/ka based on the determined adsorption rate coefficient (ka) and the dissociation rate coefficient (kd).
 3. The method according to claim 1, wherein an adsorption rate coefficient (ka) and a dissociation rate coefficient (kd) are determined based on the results of measurement of changes in surface plasmon resonance signals by using the following equations (1), (2), and (3): dθ/dt=ka×c _(s)×(1−θ)−kd×θ  (1) wherein θ represents an adsorption ratio (=amount of adsorption/amount of saturated adsorption); ka represents an adsorption rate coefficient; kd represents a dissociation rate coefficient; and c_(s) represents the concentration of the analyte molecule that exists near the metal surface; ∂c/∂t=D×∂ ² c/∂x ²   (2) wherein x represents a distance from a metal surface; D represents a diffusion coefficient of the analyte molecule; and c represents the concentration of the analyte molecule, provided that when x is 0, c is c_(s); and θ=R/R max   (3) wherein θ represents an adsorption ratio (=amount of adsorption/amount of saturated adsorption); R represents a surface plasmon signal; and Rmax represents a signal resulting when an analyte molecule is saturatedly adsorbed.
 4. The method according to claim 1, wherein the dissociation constant is determined while making corrections based on the supposition that the time required for the liquid exchange is an adsorption phenomenon under ideal conditions.
 5. The method according to claim 1, wherein the dissociation constant is determined by using nonlinear regression analysis.
 6. The method according to claim 1, which comprises using a surface plasmon resonance measurement device comprising a dielectric block, a metal film formed on one side of the dielectric block, a light source for generating a light beam, an optical system for allowing the above light beam to enter the above dielectric block so that total reflection conditions can be obtained at the interface between the dielectric block and the metal film and so that various incidence angles can be included, a flow channel system comprising a cell formed on the above metal film, and a light-detecting means for detecting the state of surface plasmon resonance by measuring the intensity of the light beam totally reflected at the above interface.
 7. The method according to claim 1 wherein the liquid contained in the above flow channel system is exchanged from a reference liquid containing no test substance to be measured to a sample liquid containing a test substance to be measured, and thereafter, a change in surface plasmon resonance is measured in a state where the flow of the sample liquid has been stopped.
 8. The method according to claim 1 wherein a reference cell, to which a physiologically active substance interacting with a test substance does not bind, is connected in series with a detection cell, to which a physiologically active substance interacting with a test substance binds, the connected cells are placed in a flow channel system, and a liquid is then fed through the reference cell and the detection cell.
 9. The method according to claim 8 wherein the ratio (Ve/Vs) of the amount of a liquid exchanged (Ve ml) in a single measurement to the volume of the above cell (Vs ml) is between 1 and
 100. 10. The method according to claim 9 wherein the ratio (Ve/Vs) is between 1 and
 50. 11. The method according to claim 1 wherein the time required for the exchange of the liquid contained in the above flow channel system is between 0.01 second and 100 seconds. 